Establishment of mouse model of inherited PIGO deficiency and therapeutic potential of AAV-based gene therapy

Inherited glycosylphosphatidylinositol (GPI) deficiency (IGD) is caused by mutations in GPI biosynthesis genes. The mechanisms of its systemic, especially neurological, symptoms are not clarified and fundamental therapy has not been established. Here, we report establishment of mouse models of IGD caused by PIGO mutations as well as development of effective gene therapy. As the clinical manifestations of IGD are systemic and lifelong lasting, we treated the mice with adeno-associated virus for homology-independent knock-in as well as extra-chromosomal expression of Pigo cDNA. Significant amelioration of neuronal phenotypes and growth defect was achieved, opening a new avenue for curing IGDs.

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Replication Randomization Blinding Full scan for western blots in Supplementary figure1 amd 4 are provided in the sourse data file. Raw data of the mouse analysis such as growth curves, blood tests and hanging tests in Figure 2 and 4 including 5'RACE of Pigo integration sites in Figure 6 and qPCR of ITR deriven Pigo expression are also provided in the source data file.
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Sample size was chosen on the bases of prior experiences or literature (Human Molecular Genetics, 2020, Vol. 29, No. 7 1205-1217, so that the biological or technical viability would be sufficiently account for.
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All experiments have been successfully repeated with similar results for at least two to three times.
For all the mouse studies, all the available and age matched KI or KIKO mice and AAV treated mice were used and the age matched wild type and hetero mice were randomly chosen for the experiments. For choosing mouse samples for MRI analysis and analysis of edited sites in the brain, we randomly chose two to four mice among the AAV treated mice. As for the analysis using knockout cells, we chose the representative clone among the several knockout clones and perform the repeated experiments using that clone.
For the scoring of severity of tremor, one investigator was blinded to pick up the mouse from the cages for the observation and determined the score together with another blinded investigator. After finishing observation of all the mice in the cage, genotype of the mice were checked. As for the seizure score, the blinded investigator watched the PTZ treated mice and determined the score.
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. For the antibodies for GPI anchored proteins, such as Gr-1, contactin-1, 2, Thy-1, CD24, CD59, they were validated by staining of GPI knockout cells compared with wild type cells in FACS, WB, and IHC. The other antibodies were validated using the isotype control. Detailed information could be found on the manufactures' web site.
Cell lines were not tested for mycoplasma.
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All mice are C57BL/6 background; Mice were maintained in SPFunder a 12-hour light/dark cycle and given free access to food and water. Temperature and humidity is within the recommended range(20~24°40~60%) No wild animals was used. This study did not involve field-collected samples.
All animal procedures were approved by the Animal Care and Use Committee of the Research Institute for Microbial Diseases, Osaka University, Japan.

March 2021
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Flow Cytometry
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Always put the isotype control staining for a negative population. Cell lines were gated with FSC/SSC for live cells. As for the mouse blood cells, lineage specific markers were used for gating.